1. Field of the Invention
This invention relates to a scanning optical microscope apparatus which is capable of detecting a plurality of fluorescent light beams at the same time.
2. Description of Related Art
In general, fluorescence photodetectors are widely used in the fields of medicine, biology, and others, for the purpose of detecting protein and genes in which living tissues and cells are labeled by fluorescence. In particular, a multiple fluorescence detecting technique that a specimen stained by a plurality of fluorescent dyes is observed at a time has recently been employed to exercise its power for the analysis of a gene and the clarification of an intra-cellular structure.
As an effective means for such fluorescence detection, a laser scanning microscope (LSM) is well known. FIG. 1 shows schematically the arrangement of the LSM for fluorescence. In this LSM, laser beams emitted from three laser oscillators 1a, 1b, and 1c which oscillate three kinds of different wavelengths are combined on a common optical path by laser-beam combination dichroic mirrors 2a and 2b. A combined laser beam is enlarged to a beam diameter of proper size through a beam expander 3, and is reflected by a dichroic mirror 4. After that, the laser beam deflected by an X-Y scanning optical system 5, such as galvanometer mirrors, and condensed through a pupil relay lens 6 and an objective lens 7 irradiates a specimen 8, which is scanned with a laser spot.
Fluorescent light from the specimen 8, excited by the irradiation of the laser beam, returns a course ranging from the objective lens 7 to the dichroic mirror 4, and after being transmitted through the dichroic mirror 4, is dispersed by a dichroic mirror 9a for dispersion. On the one hand, fluorescent light reflected by the dichroic mirror 9a is condensed by an imaging lens 10a and passes through a confocal stop 11a. After wavelengths other than that of the first fluorescent light intended are absorbed or reflected by an absorption filter 12a, its intensity is detected by a photodetector 13a. The confocal stop 11a is placed at a position optically conjugate with the focal point of the objective lens 7 and blocks light other than the fluorescent light excited by the laser spot (The same holds for confocal stops 11b and 11c). Consequently, an available image is very high in contrast. Moreover, a distance between the specimen 8 and the objective lens 7 is relatively changed along the optical axis, and thereby a three-dimensional image can be obtained.
On the other hand, fluorescent light transmitted through the dichroic mirror 9a is further dispersed by a dichroic mirror 9b. Fluorescent light reflected by the dichroic mirror 9b is condensed by an imaging lens 10b, and after passing through a confocal stop 11b, is detected in intensity by a photodetector 13b through an absorption filter 12b transmitting only the second fluorescent light intended.
Fluorescent light transmitted through the dichroic mirror 9b, after being reflected by a mirror, is condensed by an imaging lens 10c, passes through a confocal stop 11c, and is detected in intensity by a photodetector 13c through an absorption filter 12c transmitting only the third fluorescent light intended.
The LSM shown here is capable of detecting simultaneously triple excitation fluorescent light with three wavelengths emitted from the laser oscillators 1a, 1b, and 1c. Whenever the conditions of multiple excitation, such as wavelengths of laser beams, the kind of fluorescent dye, and the number of laser oscillators, are changed, the dichroic mirror 4, the dichroic mirrors 9a and 9b for dispersion, and the absorption filters 12a, 12b, and 12c are replaced with those having the optimum dispersing characteristics.
However, a conventional LSM for fluorescence using optical filters has the following problems. First, an optical filer is such that its dispersing characteristic cannot be determined at will because of restrictions on fabrication, and thus the amount of fluorescent light and an S/N ratio are limited. In particular, the absorption filter needs to completely block excited light, but at present cannot be designed or fabricated so as not to loss the amount of light in the wavelength region of the highest fluorescence intensity, close to the wavelength of excited light. Second, expensive optical filters which are exclusively used in accordance with the wavelength of excited light and the fluorescent dye must be prepared, and when a variety of multiple excitation are assumed, it is unavoidable to cause an increase in the number of filters and the complication and oversizing of an apparatus. Third, as will be obvious from the LSM for fluorescence shown in FIG. 1, the multiple fluorescence is dispersed through a plurality of optical filters, and hence a considerable amount of light is lost before fluorescent light reaches each of the photodetectors. Any of these problems becomes severe as the multiplicity of excited light and fluorescent light increases.
In order to solve the above problems, several techniques of selecting and detecting a plurality of fluorescence wavelengths without using the optical filters are proposed. For example, WO 95/07447 discloses a spectroscope and a confocal fluorescence microscope in which a light beam decomposed into a wavelength spectrum by a prism is dispersed into a first wavelength region transmitted and a second wavelength region reflected by a slit-like mirror, and the position and width of a second slit restricting the slit-like mirror and the second wavelength region are controlled so that two arbitrary wavelength regions can be selected and detected.
On the other hand, Japanese Patent Preliminary Publication No. Hei 8-43739 discloses a scanning optical microscope in which a light beam passing through a confocal stop is dispersed by a grating so that a wavelength region and a wavelength width are selected by at least one slit, and the amount of light in the wavelength region is detected by a photodetector.
In the fluorescence detection for multiple excitation, each of these techniques is a means for providing a scanning optical microscope which surely blocks excitation wavelengths without using the optical filters and holds a sufficient amount of fluorescence to make the fluorescence detection with a high S/N ratio.
A spectrometer which is capable of selecting any wavelength without using the optical filters is disclosed, for example, in U.S. Pat. No. 5,504,575. This device is such that after a light beam to be detected is spatially decomposed into a wavelength spectrum by a dispersion element, at least one part of a dispersion spectrum is received by a spatial light modulator represented by a deformable mirror device, and only light in a desired spectral region is reflected or transmitted to detect its energy intensity. Once the relation between the dispersion element and the spatial light modulator or an energy detector is established, any mechanical movement, except for the spatial light modulator, is not required, and thus an error can be eliminated. Furthermore, a high-precision mechanical control becomes unnecessary.
The details of the deformable mirror device are set forth in U.S. Pat. No. 5,061,049. Specifically, this device includes a spatial array of micromirrors, each of which is capable of deflecting light at an arbitrary angle previously selected, only by the control of an applied voltage.
However, in WO 95/07447 mentioned above, slits determining a wavelength selection are attended with mechanical movements and their operating section requires an extremely high-grade control structure. Moreover, correction is required because the wear and vibration of a mechanical drive impair the reproducibility of measurement. In particular, when the light beam is dispersed, the slit-like mirror and the second slit must be mutually associated to move, and therefore it is very difficult to control this movement with a high degree of accuracy. In addition, if the kinds of fluorescence to be detected simultaneously are increased, one dispersion means is unsatisfactory, and a plurality of wavelength separating means must be used, thus causing a loss in the amount of light and the complication of the device.
In Hei 8-43739, as in the case of the scanning optical microscope in the foregoing, the accuracy of the mechanical drive and the reproducibility must be ensured. In addition to this problem, since a wavelength band which can be detected by a single photodetector is determined by a spectrum dispersing means such as a grating and the initial placement of the photodetector, the number of degrees of wavelength selection freedom in a broad wavelength band is too small to accommodate the varieties of the fluorescent dye and the laser beam wavelength in the fluorescence detection for multiple excitation.
The spectrometer disclosed in U.S. Pat. No. 5,504,575 is such that the detection of the amount of light in an arbitrary wavelength region is possible, and thus where one fluorescent light beam is obtained with respect to one excitation wavelength, this device can be applied to the LSM for fluorescence. However, where the intensities of fluorescent light multiplied by accommodating any combination of the excitation wavelength and the fluorescent dye are detected at the same time, the device cannot be easily incorporated in the LSM for fluorescence.
It is, therefore, an object of the present invention to provide a scanning optical microscope apparatus which has a simple structure in which optical filters are not used and a mechanical drive requiring a high degree of accuracy in positional reproducibility need not be provided, and is capable of detecting a fluorescent image for multiple excitation of a multiply stained specimen with a high S/N ratio, without changing its arrangement in various combinations of the excitation wavelength and the fluorescence dye.
In order to achieve the above object, the scanning optical microscope apparatus of the present invention includes a laser scanning optical microscope apparatus provided with a laser light source, an objective lens for condensing a laser beam emitted from the laser light source on a specimen, a scanning means for relatively scanning the specimen with a condensed laser spot, an imaging optical system for imaging light emanating from the specimen, a confocal stop placed at the focal point of the imaging optical system, and a plurality of photodetectors for detecting the light from the specimen, passing through the confocal stop. In this case, the scanning optical microscope apparatus includes a spectrum decomposing means for spatially decomposing a light beam passing through the confocal stop into a wavelength spectrum, and an array of light-deflecting microelements arranged, at least, in a direction of spectral decomposition and receiving and deflecting a part of the light beam decomposed into the spectrum toward any of the plurality of photodetectors. Each of the light-deflecting microelements has a plurality of deflection angles at which the light beam is selectively received by any of the plurality of photodetectors so that one of the plurality of deflection angles can be selected at will.
The scanning optical microscope apparatus satisfies the following condition:
d/xcex4xcex less than 0.2
where d is the dimension of each of the light-deflecting microelements in the direction of spectral decomposition and xcex4xcex is a distance between positions on the array of light-deflecting microelements on which two wavelengths of 656.27 nm and 486.13 nm, separated by the spectrum decomposing means, are incident.
Further, the scanning optical microscope apparatus is designed so that a laser beam for excitation is oscillated to thereby detect the position of each light-deflecting microelement corresponding to the wavelength of the laser beam, and the deflection angle of each light-deflecting microelement is determined in accordance with the information of this detection.
This and other objects as well as the features and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments when taken in conjunction with the accompanying drawings.